Our objective is to gain deeper insight into electron transport in heart muscle by studying and comparing heart mitochondria and a bacterium chosen for its relationship to mammalian mitochondria. We will investigate the respiratory chain of the inner mitochondrial membrane of mammalian heart muscle and the cytoplasmic membrane of the aerobic bacterium Paracoccus denitrificans with techniques we have developed. We will focus on the functional and structural aspects of the reactions by which cytochrome c transports electrons from cytochrome c reductase to cytochrone c oxidase. We will continue to study the conditions needed for the formation of our new, high-turnover, especially reactive, complex of cytochrome c-cytochrome oxidase that has either increased interaction between the components or the capacity for more rapid reduction or oxidation of the proper components. We will examine the experimental conditions which are required for the appearance of a similar reactive complex of cytochrome c-cytochrome c reductase. Since the variety of assay procedures for cytochrome oxidase that have been employed by different laboratories have resulted in determinations not comparable or interpretable, we will work to establish a uniform acceptable method of relating spectrophotometric and polarographic assays. Our laboratory is the first to develop monoclonal antibodies to cytochrome c. Using hybridoma cell lines that we have generated, we have produced 15 different exquisitely specific monoclonal antibodies to both beef cytochrome c and P. denitrificans cytochrome c. Some of these antibodies bind only the homologous antigen and some are cross reactive. We will utilize this library of monoclonal antibodies which we are continuing to develop, to probe sites on the cytochrome c which are involved in its interactions with oxidases, reductases and peroxidases in mammalian and bacterial electron transport systems. We will perform competition binding assays to determine the number of distinct epitopes (antigenic sites) recognized by these homogeneous antibodies. Studies will be aimed at the electron transport chain of P. denitrificans which is of particular interest because it is so similar in composition to the chain of the mitochondrial inner membrane, and may be its evolutionary precursor. Other advantages of this organism are the ease with which subcellular particles can be isolated, and the possibility of varying components of the chain by changing the growth conditions.